Lab 7

Lab 7: Pre-Lab

Your task in Lab 7 is to carryout your experiment regarding the rate of cellular respiration and begin to analyze your data. To prepare for Lab 7, please review this pre-lab page. Once you feel confident regarding the below topics, complete the corresponding LABridge in Blackboard.

Introduction/Review
Do you know enough?
What will we do in lab?
LABridge

Cellular Respiration

CONNECTION ALERT! Cellular respiration is the topic of Chapter 9 in your BIOL 120 lecture. Please review your textbook as needed for this lab.

Energy is the currency of life: all living organisms require energy to survive and reproduce. Metabolism is the series of reactions and processes, catalyzed by enzymes, which together maintain life. These reactions fall into two types: catabolic or anabolic. These processes are the inverse of each other and in photosynthetic organisms occur in tandem as the anabolic reactions of photosynthesis create the products that are then broken down by the catabolic reactions of cellular respiration (view figure at left).

There are two general classes of cellular respiration that are characterized by their relative efficiency (ATP production): anaerobic (without oxygen) and aerobic (oxygenated) respiration. We are focusing on aerobic respiration in this lab, which is a highly efficient process occurring within the mitochondria of eukaryotic organisms that have higher energy requirements for survival. In a 4 step process, oxygen and glucose are used to produce energy (ATP), H2O, and CO2,

Glycolysis ("splitting of sugar"): This step happens in the cytoplasm. One Glucose (C6H12O6) is broken down to 2 molecules of pyruvate. Requires 2 ATP to start, produces 4 with a 2 ATP net payoff.
Pyruvate Grooming: The pyruvate from glycolysis is shuttled into the mitochondria, where it is converted to a molecule called Acetyl CoA for further breakdown.
The Citric Acid Cycle: Occurs in the mitochondrial matrix. In the presence of oxygen (O2), all the hydrogens (H2) are stripped off the Acetyl CoA, two by two, to extract the electrons for making ATP, until there are no hydrogens left - and all that is left of the sugar is CO2 - a waste product - and H2O. The Citric Acid Cycle results in the production of only ~4 ATPs, but produces a lot of NADH, which will go on to the next step.
The Electron Transport Chain and Chemiosmosis ("the big ATP payoff"). Occurs in the christae of the mirochondria, the folded membranes inside. Electrons from hydrogen are carried by NADH and passed down an electron transport chain. The energy release runs pumps that creat an electrochemical gradient of H+ ions. In Chemiosmosis, a big pump called ATP Synthase uses the gradient to produce A LOT of ATP. Results in the production of ~28-32 ATPs for every molecule of glucose.

Review the steps of anaerobic and aerobic respiration by clicking the links in the sidebar.

Be sure you know and understand the generalized equation for aerobic cellular respiration.

ANAEROBIC Respiration review

Aerobic respiration review

Do you know enough about crayfish?

-From the Kentucky Wildlife Action Plan (WAP)

Abundance. There are 640 species of freshwater crayfish (Order Decapoda) worldwide (Crandall and Buhay 2008). More than 360 species are in the US and the southeast is known as a hotspot of crayfish diversity (Taylor et al. 2007). Kentucky is inhabited by 54 species, with some under taxonomic review and others potentially awaiting discovery. Seven species are endemic to the state of Kentucky.

Status. Freshwater crayfish are valuable indicator species of watershed health. They act as "canaries in the coal mine" and provide early warnings of ecosystem distress when populations decline. Modification of habitats, sedimentation, and dams are serious threats to freshwater crayfishes. Nationally, about 48% of crayfish species are of conservation concern (ranging from Vulnerable to Endangered); over a third (37%) of the Kentucky fauna falls into this category (KSNPC, 2010). Cave species are particularly at-risk from upland activities that pollute groundwater flowing into cave systems; this includes issues with chemical spills, agricultural runoff, salt from roads, and siltation from poor land use.

Review the links in the sidebar: Facts on KY Crayfish and Crayfish Natural History. Also, watch the short video below to see these guys in action!

Facts on KY Crayfish

Crayfish Natural History

What will we do in lab?

Your task was to design a proposal in Lab 6. In lab 7, we will conduct an experiment that tests the affects of one variable on the rate of cellular respiration in crayfish (AKA craw-fish or crawl-dad). Like most animals, they rely on aerobic cellular respiration to meet their energy demands.

Review the general procedural steps we will use below. Your exact methods will differ depending on which variable you have selected to test. Make sure you are comfortable with concepts below.

How will we measure the rate of cellular respiration? ...By measuring the CO2 produced as a bi-product of the metabolic process! You will place your crayfish in an aquatic environment and allow it to "respire." After a set period of time (decided by you). Then you will remove the crayfish and analyzed how much CO2 was produced as a result of cellular respiration.
Review the following steps to understand how we will quantify the respiration rate.
The water you will place your crayfish will be slightly basic and will appear pink! This is because it will contain a pH indicator called, phenolphthalein, which is pink in the presence of a base and turns clear when it approaches neutral (pH=7).
As cellular respiration is occurring inside your crayfish , inside the beaker, CO2 will be expelled into the solution as a bi-product. In an aqueous environment, this CO2 breaks down into carbonic acid (H2CO3) then into bicarbonate (HCO3-) and H+ ions.
Increased concentrations of H+ ions increase the acidity in solution and lower the pH. As the pH decreases the solution will turn from pink (basic) to clear (neutral).
BUT! We will not wait for that to happen on its own, it would take far too long!
So...after your crayfish "respires" in solution for your set period time, you will remove it. The water will still appear pink. Some respiration and CO2 production HAS occurred, but not enough to change the pH to see a color change. So we would add more acid to the solution (H2SO4), via titration, until we see a color change from pick to clear. View Diagram.
You will use the "amount of titrant used" to calculate the micromoles of carbonic acid (H2CO3) produced in solution using this equation.
Lastly you will complete your analysis using group or class data including creating a bar graph and using an unpaired t-test calculator to test your hypothesis.

Review the YouTube video below on titration with phenollphthalein so you have a good idea of what to expect in lab!

How pH change indicates respiration rate. Click to enlarge.

Special Note! You have no doubt noticed the importance of sample size for this experiment to work well. Depending on your lab size, timing, and proposals, you may be asked to share data as a class or among several groups testing the same variables. You may also be asked to pick just one variable and all test it together as a class.

Be sure you understand the relationship between the amount of titrant required and the rate of cellular respiration.

Click here to get to WKU's blackboard to take your LABridge for this week. Be sure your Notebook Entry from last lab is ready to submit!

Lab 7: Protocol

In today's lab you will work with your lab group to conduct your experiment and begin to analyze your data.

Exercise I. Review your research proposal and the pre-lab and revise where necessary

Exercise II. Conduct your experiment & collect data

Exercise III. Analyze your data

Lab Objectives: Following today's lab, you should be able to...

Exercise I
Exercise II
Exercise III

Exercise I. Prepare

Procedure

Open the Lab 7 Notebook Guide
Open your research proposal on Cellular Respiration from Lab 6.
Review the pre-lab.
At this point your class might decide to work together to narrow down your choices. If you work together as a class in large groups, you can increase your sample size.
Review the slide show below (from Lab 6) for a refresher on the parameters in your experimental protocol as needed.
Complete Exercise I in the the Lab Notebook Guide. Ask the following questions and augment/revise your proposal as necessary.
1. Hypotheses remain the same?
2. Independent, dependent and confounding variables remain the same?
3. How long does your crayfish need to respire?
4. How many trials will you do?
5. Do you need a control? (YES) ...depending on your experiment. What should it be?
6. What kind of statistics will you use?
7. What kind of graph might you make?
STOP. You must get your instructor's "OK" before you can move-on. She/he will need to go over your Lab Notebook Guide before you proceed.

Lab 7 Notebook Guide. Click to download.

Exercise II. Run your experiment & collect data

Gloves and goggles all day.
Sulfuric acid is dilute but requires caution.
Shellfish allergies should be reported.
Any cruel treatment of animals in BIOL 121 will not be tolerated. Be careful. Be respectful.

Any cruel treatment of animals in BIOL 121 will not be tolerated. Be careful. Be respectful.
Do NOT titrate your crayfish.
Do NOT put your crayfish on its back.

Decide who will be responsible for what. Refer to your contract.
You can titrate controls while your treatments with crayfish are running. You can run multiple trials at the same time. You'll need a careful and responsible timer and a slow and steady titration team.

Remember: Basic solutions are pink with the phenolphthalein indicator and acidic solutions are clear with the phenolphthalein indicator.
How much "work" (cellular respiration) did the crayfish due while in solution? We can determine that through titrations. We will add drops of an acidic titrant (sulphuric acid) until the solution reaches a clear endpoint. If we need to add lots of drops, then the solution was farther from the endpoint because the crayfish did not expel much CO2 and must have had a lower rate of cellular respiration. If we need to add fewer drops, then the solution was closer to the endpoint because crayfish released alot of CO2 and must have had a higher rate of cellular respiration.

Generalized Procedure: Yours will vary based on your own protocol and variables

At minimum, you must test one crayfish in condition 1 and one crayfish in condition 2. If you have additional time you can add multiple trials.
*Items in pink below can vary based on the size of our crayfish each term and will be provided by the TA.

Gather your supplies and review Exercise II in the Lab Notebook Guide.
Open your data collection spreadsheet (sidebar). Your TA will provide the values for the Nr variable.
Obtain a pitcher of your stock solution.
Fill your beaker depending on the first variable you will test.
Obtain your crayfish and determine it's volume. Size matters here. Some terms our crayfish are larger than others. Follow your TA's instructions on the volume required.
Following your own protocol, place your crayfish in its test environment of stock solution and begin your test.
While you wait for your crayfish to respire: PRACTICE YOUR TITRTATIONS (see directions in sidebar). Your TA will demo this procedure or help you each group by group when you are ready. The directions are in the sidebar.
Record notes and data in your spreadsheet as you go along.
The amount of time needed for your crayfish to "respire" can vary by size. Your TA will provide the minimum each term.
After your allotted time, remove your crayfish and place it (gently) in the recovery tank on the front desk.
Conduct your acid-base titration.
You will use 50 mL of your experimental solution for the titration.
Record your titration results in your excel spreadsheet.
At this point, you can start your second test following the same directions as above.
If you have now tested both conditions with at least one crayfish, you can move on to the next step, or continue testing to add more trials based on the time available.
Use this equation to calculate to umols of carbonic acid produced by your crayfish. The Nr values are available on the board for each condition.
Once you have completed the calculations add your data to the class or group data on the board.
Open the second tab in your Excel spreadsheet labeled "Class or Group data."
Enter in the class or group data from the board into each column. Remember these values are in umol of carbonic acid produced by the crayfish in two groups (i.e., in the two different treatment conditions).
Complete Exercise II in your Lab Notebook Guide.

REMEMBER why we have to use titration! In an aqueous environment, CO2 combines with water to first create carbonic acid, which is then broken down into hydrogen ions. The addition of H+ ions results in a change in pH: the pH decreases and becomes more acidic. The introduction of CO2 from the crayfish would have lowered the pH some, but not enough in 20 minutes to make it acidic enough to turn clear.

How to determine crayfish volume. Click to enlarge. Use 50mL for F23.

Practice directions.

Titration directions. Click to enlarge.

Lab 7 data collection excel sheet. Click to download.

Exercise III. Analysis

Procedure

Now, what type of statistics should we use to determine if the mean respiration rate between our 2 groups is significantly different? ...hopefully this is an easy answer. Are the mean umols carbonic acid (H2CO3) produced in your two groups the same or are they different?
Use the tools in the sidebar to complete your Lab Notebook Guide. You will need to create a bar graph in Excel, run a T-test and answer a set of questions.

Remember: Statistics solve the problem of determining if "more" or "higher" or "different" than is actually enough to be important and biological relevant. Using the principles of probability, they help us parse what we observe from randomness (chance alone) as meaning (a real difference, or a real relationship). Statistics tell us how likely we would be to make the same observations we have made, if chance and randomness were the only drivers. If the probability is very low (<5%), we refer to these patterns as significant.

Visit the stats section of our research library.

Unpaired Bar Graph Demo

unpaired t-test CALCULATOR demo

T-test Online Calculator.

How big can they get? Check this out...our very own Dr. Huskey!

Lab 7 BIOL 120 CONNECTIONS
Section 1.6: Doing Biology
Big Picture 1: How to Think Like a Scientist
BioSkillls 2: Reading & Making Graphs
BioSkillls 3: Interpreting Standard Error and Using Statistical Tests
BioSkillls 4: Working with Probabilities
Chapter 9: Cellular Respiration

Faculty Spotlight: Dr. Noah Ashley

noah.ashley@wku.edu

We are conducting experiments to identify factors that affect the physiological process of cellular respiration in crayfish. Similarly, Dr. Noah Ashley's lab works to identify physiological, immunological, and behavioral responses to various factors, like sickness and sleep loss, in mice and birds. Specifically, they are investigating the costs and benefits of the sickness response in vertebrates, the inflammatory response in sleep-deprived mice, sleep loss in migratory birds, and the sleep-wake cycle in arctic songbirds. Dr. Ashley's lab is extremely productive! His research proposals have been funded by the NSF and the NIH. Learn more here (Lab Web Page). You just might recognize one of his current graduate students!

Research Key Words: physiology, ornithology, eco-immunology, genomics, sleep loss, arctic song birds, migrating birds
Recent Publication: Cooper, L. N., Mishra, I., Ashley, N.T. 2019. Short-Term Sleep Loss Alters Cytokine Gene Expression in Brain and Peripheral Tissues and Increases Plasma Corticosterone of Zebra Finch (Taeniopygia guttata). Physiological and Biochemical Zoology 92:80-91.

​[Metabolism]What factors affect the rate of cellular respiration in crayfish?